Bacteroides are gram negative obligately anaerobic bacteria that account for 25% of the complex microbial population that resides in the normal human colon. These bacteria are notable for their ability to utilize a variety of polysaccharides, and they may be responsible for the extensive digestion of dietary and host polysaccharides that occurs in the colon. Bacteroides are also the main cause of opportunistic anaerobic infections in humans, and their ability to degrade tissue polysaccharides may contribute to their virulence. Bacteroides polysaccharide-degrading enzymes are generally not extracellular, and there appear to be outer membrane proteins that bind polysaccharides and facilitate their passage through the outer membrane. We will use biochemical and genetic techniques to investigate the steps involved in catabolism of polysaccharides by Bacteroides. We will clone polysaccharide utilization genes by complementing mutations that affect early steps in catabolism and use these clones to determine the number and organization of genes needed for catabolism. We will use gene disruption techniques to assess the contribution of various polysaccharides and membrane proteins to the degradative process. We will also use cloned genes and regulatory mutants to determine how polysaccharide utilization genes are regulated. Information obtained in these pure culture studies will be used to develop methods for determining what Bacteroides are actually doing in the colon. Specifically, we will use antiserum that detects polysaccharide-regulated proteins to determine whether these proteins are expressed at induced levels in human feces. We will also use germfree mice as a model system for testing the effect of mutations in intestinal tract. The results of these experiments should provide new information about the activities of the human colonic microflora and how these activities are affected by the host's diet.